High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

Microbially synthesized modular virus-like particles and capsomeres displaying group A streptococcus hypervariable antigenic determinants.

Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 10(4) ). Second, delivery by capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 µg (corresponding to <600 ng of each GAS peptide), induced significant titers of IgGs specific to each peptide, demonstrating that a multivalent, broad-coverage VLP vaccine formulation was possible. In summary, the modular VLPs and capsomeres reported here demonstrate, with promising preliminary data, innovative ways to design GAS vaccines using VLP and capsomere delivery systems amenable to microbial synthesis, potentially adoptable by developing countries.

Safety and immunogenicity of a Sf9 insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine.

OBJECTIVE: We performed a Phase 1 randomized, observer-blinded, placebo-controlled trial to evaluate the safety and immunogenicity of a recombinant respiratory syncytial virus (RSV) fusion (F) protein nanoparticle vaccine. METHODS: Six formulations with (5, 15, 30 and 60 µg) and without (30 and 60 µg) aluminum phosphate (AdjuPhos) were administered intramuscularly on day 0 and 30 in a dose escalating fashion to healthy adults 18-49 years of age. Solicited and unsolicited events were collected through day 210. Immunogenicity measures taken at day 0, 30 and 60 included RSV A and B microneutralization, anti-F IgG, antigenic site II peptide and palivizumab competitive antibodies. RESULTS: The vaccine was well-tolerated, with no evident dose-related toxicity or attributable SAEs. At day 60 both RSV A and B microneutralization was significantly increased in vaccinees versus placebo. Across all vaccinees there was a 7- to 19-fold increase in the anti-F IgG and a 7- to 24-fold increase in the antigenic site II binding and palivizumab competitive antibodies. CONCLUSIONS: The RSV F nanoparticle vaccine candidate was well tolerated without dose-related increases in adverse events. Measures of immunity indicate that neutralization, anti-RSV F IgG titers and palivizumab competing antibodies were induced at levels that have been associated with decreased risk of hospitalization. NCT01290419.

Long-term and memory immune responses in mice against Newcastle disease virus-like particles containing respiratory syncytial virus glycoprotein ectodomains.

Although respiratory syncytial virus (RSV) is a significant human pathogen, no RSV vaccines are available. We have reported that a virus-like particle (VLP) RSV vaccine candidate stimulated, in mice, robust, protective anti-RSV glycoprotein T(H)1 biased immune responses without enhanced respiratory disease upon RSV challenge. We report here an analysis of long-term responses to these VLPs. BALB/c mice immunized, without adjuvant, with VLPs or with infectious RSV generated anti-F and anti-G protein serum antibody responses that were stable over 14 months. Neutralizing antibody titers stimulated by VLPs were robust and durable for 14 months, whereas those of RSV-immunized animals declined significantly by 3 months. F protein-specific antibody-secreting cells were detected in the bone marrows of VLP-immunized mice but not in the marrows of RSV-immunized mice. Adoptive transfer of enriched splenic B cells from VLP-immunized mice into immunodeficient rag(-/-) mice resulted in anti-F and anti-G protein serum IgG antibody responses, in recipient mice, that were protective upon RSV challenge. In contrast, transfer of splenic B cells from RSV-immunized mice produced no detectable serum antibody in the recipients, nor could these mice inhibit RSV replication upon virus challenge. Immunization with VLPs stimulated the formation of germinal center GL7(+) B cells in normal mice. VLP immunization of TCR ßδ(-/-) T-cell-deficient mice did not induce anti-RSV IgG antibodies, results consistent with T-cell-dependent immune responses. These results demonstrate that VLPs are effective in stimulating long-lived RSV-specific, T-cell-dependent neutralizing antibody-secreting cells and RSV-specific memory responses.

Influenza virosome/DNA vaccine complex as a new formulation to induce intra-subtypic protection against influenza virus challenge.

Influenza virosome is one of the commercially available vaccines that have been used for a number of years. Like other influenza vaccines, the efficacy of the virosomal vaccine is significantly compromised when circulating viruses do not have a good match with vaccine strains due to antigenic drift or less frequent emergence of a pandemic virus. A major advantage of virosome over other influenza vaccine platforms is its intrinsic adjuvant activity and potential carrier capability which have been exploited in this study to broaden vaccine protectivity by incorporating a conserved component of influenza virus in seasonal vaccine formulation. Influenza nucleoprotein (NP)-encoding plasmid was adsorbed onto surface of influenza virosomes as a virosome/DNA vaccine complex. Mice were immunized with a single dose of the influenza virosome attached with the NP plasmid or NP plasmid alone where both influenza virosomes and NP gene were derived from influenza A virus H1N1 New/Caledonia strain. Analysis of the cellular immune responses showed that 5µg (10-fold reduced dose) of the NP plasmid attached to the virosomes induced T cell responses equivalent to those elicited by 50µg of NP plasmid alone as assessed by IFN-γ and granzyme B ELISPOT. Furthermore, the influenza virosome/NP plasmid complex protected mice against intra-subtypic challenge with the mouse adapted H1N1 PR8 virus, while mice immunized with the virosome alone did not survive. Results of hemagglutination inhibition test showed that the observed intra-subtypic cross-protection could not be attributed to neutralizing antibodies. These findings suggest that influenza virosomes could be equipped with an NP-encoding plasmid in a dose-sparing fashion to elicit anti-influenza cytotoxic immune responses and broaden the vaccine coverage against antigenic drift.

Enhanced influenza virus-like particle vaccines containing the extracellular domain of matrix protein 2 and a Toll-like receptor ligand.

The extracellular domain of matrix protein 2 (M2e) is conserved among influenza A viruses. The goal of this project is to develop enhanced influenza vaccines with broad protective efficacy using the M2e antigen. We designed a membrane-anchored fusion protein by replacing the hyperimmunogenic region of Salmonella enterica serovar Typhimurium flagellin (FliC) with four repeats of M2e (4.M2e-tFliC) and fusing it to a membrane anchor from influenza virus hemagglutinin (HA). The fusion protein was incorporated into influenza virus M1-based virus-like particles (VLPs). These VLPs retained Toll-like receptor 5 (TLR5) agonist activity comparable to that of soluble FliC. Mice immunized with the VLPs by either intramuscular or intranasal immunization showed high levels of systemic M2-specific antibody responses compared to the responses to soluble 4.M2e protein. High mucosal antibody titers were also induced in intranasally immunized mice. All intranasally immunized mice survived lethal challenges with live virus, while intramuscularly immunized mice showed only partial protection, revealing better protection by the intranasal route. These results indicate that a combination of M2e antigens and TLR ligand adjuvants in VLPs has potential for development of a broadly protective influenza A virus vaccine.

Developments of subunit and VLP vaccines against influenza A virus.

Influenza virus is a continuous and severe global threat to mankind. The continuously re-emerging disease gives rise to thousands of deaths and enormous economic losses each year, which emphasizes the urgency and necessity to develop high-quality influenza vaccines in a safer, more efficient and economic way. The influenza subunit and VLP vaccines, taking the advantage of recombinant DNA technologies and expression system platforms, can be produced in such an ideal way. This review summarized the recent advancements in the research and development of influenza subunit and VLP vaccines based on the recombinant expression of hemagglutinin antigen (HA), neuraminidase antigen (NA), Matrix 2 protein (M2) and nucleocapsid protein (NP). It would help to get insight into the current stage of influenza vaccines, and suggest the future design and development of novel influenza vaccines.

Comparison of the immune responses to the CIA06-adjuvanted human papillomavirus L1 VLP vaccine with those against the licensed HPV vaccine Cervarix™ in mice.

CIA05 is a toll-like receptor (TLR) 4 agonist derived from an Escherichia coli lipopolysaccharide (LPS) mutant and has been shown to have potential as a vaccine adjuvant. In this study, we investigated the immunopotentiating activity of the adjuvant system CIA06, which is comprised of CIA05 and aluminum hydroxide (alum), when used with the human papillomavirus (HPV) L1 virus-like particles (VLPs) vaccine. BALB/c mice were immunized intramuscularly three times at 2-week intervals with HPV16 L1 VLPs alone or in the presence of various combinations of CIA05 and alum, and the immune responses were assessed. We found that the combination of CIA05 and alum at a ratio of 1:50 (designated CIA06B) yielded the highest immune response in terms of serum anti-HPV L1 VLP IgG antibody titers, splenocyte interferon (IFN)-γ secretion, and antigen-specific memory B cell responses. The immunogenicity of the CIA06B-adjuvanted HPV16/18 L1 VLP vaccine was compared with that of the currently licensed HPV vaccine Cervarix™. The CIA06B-adjuvanted vaccine was similar to Cervarix™ with regard to eliciting serum antigen-specific IgG antibodies and virus-neutralizing antibodies but more effective at inducing splenic cytokine production and memory B cells. We also observed that the antigen-specific IgG antibody titers, splenic IFN-γ secretion and memory B cells induced by the CIA06B-adjuvanted HPV vaccine remained high up to 24 weeks post-immunization. Based on these data, we concluded that CIA06B may have potential as an adjuvant in a potent prophylactic vaccine against HPV infection.

Immunogenicity and specificity of norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations.

Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 "Consensus" VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH)(3)) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.

Production and characterization of mammalian virus-like particles from modified vaccinia virus Ankara vectors expressing influenza H5N1 hemagglutinin and neuraminidase.

Several studies have described the production of influenza virus-like particles (VLP) using a variety of platform systems. These VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins and have been proposed as vaccine candidates for both seasonal and pandemic influenza. Although still in the early stages of development and evaluation as influenza vaccines, influenza VLPs have a variety of other valuable uses such as examining and understanding correlates of protection against influenza and investigating virus-cell interactions. The most common production system for influenza VLPs is the baculovirus-insect cell expression which has several attractive features including the ease in which new gene combinations can be constructed, the immunogenicity elicited and protection afforded by the produced VLPs, and the scalability offered by the system. However, there are differences between the influenza VLPs produced by baculovirus expression systems in insect cells and the influenza viruses produced for use as current vaccines or the virus produced during a productive clinical infection. We describe here the development of a modified vaccinia virus Ankara (MVA) system to generate mammalian influenza VLPs containing influenza H5N1 proteins. The MVA vector system is flexible for manipulating and generating various VLP constructs, expresses high level of influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins, and can be scaled up to produce VLPs in quantities sufficient for in vivo studies. We show that mammalian VLPs are generated from recombinant MVA vectors expressing H5N1 HA alone, but that increased VLP production can be achieved if NA is co-expressed. These mammalian H5N1 influenza VLPs have properties in common with live virus, as shown by electron microscopy analysis, their ability to hemagglutinate red blood cells, express neuraminidase activity, and to bind influenza specific antibodies. Importantly, these VLPs are able to elicit a protective immune response in a mouse challenge model, suggesting their utility in dissecting the correlates of immunity in such models. Mammalian derived VLPs may also provide a useful tool for studying virus-cell interactions and may have potential for development as pandemic vaccines.

Virus-like particles of hepatitis B virus core protein containing five mimotopes of infectious bursal disease virus (IBDV) protect chickens against IBDV.

Current infectious bursal disease virus (IBDV) vaccines suffer from maternal antibody interference and mimotope vaccines might be an alternative. Previously we demonstrated an IBDV VP2 five-mimotope polypeptide, 5EPIS, elicited protective immunity in chickens. In the current study, the 5epis gene was inserted into a plasmid carrying human hepatitis B virus core protein (HBc) gene at its major immunodominant region site. The recombinant gene was efficiently expressed in Escherichia coli to produce chimeric protein HBc-5EPIS which self-assembles to virus-like particles (VLP). Two-week old specific-pathogen-free chickens were immunized intramuscularly with HBc-5EPIS VLP or 5EPIS polypeptide without adjuvant (50 µg/injection) on day 0, 7, 14 and 21. Anti-5EPIS antibody was first detected on day 7 and day 21 in HBc-5EPIS and 5EPIS groups, respectively; on day 28, anti-5EPIS titers reached 12,800 or 1600 by ELISA, and 3200 or 800 by virus neutralization assay in HBc-5EPIS and 5EPIS groups, respectively. No anti-5EPIS antibody was detected in the buffer control group throughout the experiment. Challenge on day 28 with a virulent IBDV strain (GX8/99) resulted in 100%, 40.0% and 26.7% survival for chickens immunized with HBc-5EPIS, 5EPIS and buffer, respectively. These data suggest epitope presentation on chimeric VLP is a promising approach for improving mimotope vaccines for IBDV.

Protective efficacy of Bluetongue virus-like and subvirus-like particles in sheep: presence of the serotype-specific VP2, independent of its geographic lineage, is essential for protection.

There have been multiple separate outbreaks of Bluetongue (BT) disease of ruminants in Europe since 1998, often entering via the Mediterranean countries of Italy, Spain and Greece. BT is caused by an orbivirus, Bluetongue virus (BTV), a member of the family Reoviridae. BTV is a non-enveloped double-capsid virus, which encodes 7 structural proteins (VP1-VP7) and several non-structural proteins (NS1, NS2, NS3/3a and NS4) from ten double-stranded RNA segments of the genome. In this report, we have prepared BTV virus-like particles (VLPs, composed of VP2, VP3, VP5 and VP7) and sub-viral, inner core-like particles (CLPs, VP3 and VP7) using a recombinant baculovirus expression system. We compared the protective efficacy of VLPs and CLPs in sheep and investigated the importance of geographical lineages of BTV in the development of vaccines. The Greek crossbred Karagouniko sheep, which display mild to sub-clinical BT, were vaccinated with VLPs or CLPs of BTV-1, derived from western lineage and were challenged with virulent BTV-1 from an eastern lineage. All VLP-vaccinated animals developed a neutralising antibody response to BTV-1 from both lineages prior to challenge. Moreover, post-challenged animals had no clinical manifestation or viraemia and the challenged virus replication was completely inhibited. In contrast, CLP-vaccinated animals did not induce any neutralising antibody response but developed the group specific VP7 antibodies. CLPs also failed to prevent the clinical manifestation and virus replication, but in comparison to controls, the severity of disease manifestation and viraemia was mitigated. The data demonstrated that the outer capsid was essential for complete protection, while the geographical origin of the BTV was not critical for development of a serotype specific vaccine.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

BACKGROUND: The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. METHODS: To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. RESULTS: Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. CONCLUSION: Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

Induction of insert-specific immune response in mice by hamster polyomavirus VP1 derived virus-like particles carrying LCMV GP33 CTL epitope.

Hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) carrying model GP33 CTL epitope derived from Lymphocytic choriomeningitis virus (LCMV) were generated in yeast and examined for their capability to induce CTL response in mice. Chimeric VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo and induced antigen-specific CD8+ T cell proliferation. Mice immunized only once with VP1-GP33 VLPs without adjuvant developed an effective GP33-specific memory T cell response: 70% were fully and 30% partially protected from LCMV infection. Moreover, aggressive growth of tumors expressing GP33 was significantly delayed in these mice in vivo. Therefore, HaPyV VP1-derived VLP harboring CTL epitopes are attractive vaccine candidates for the induction of insert-specific CTL immune response.

[Advantages and prospects of the use of genetic vaccines for the protection from dangerous and socially significant infections].

High frequency of epidemiological threats (H5N1 influenza, SARS, etc.) in modern world calls for the development of new flexible technologies for manufacturing efficacious vaccines and rapid reorientation of their production as appropriate. Genetic vaccination is one of such technologies aimed at prophylaxis of dangerous and socially significant infections. The technology is based on administration of one or several functionally active genes encoding for antigens of pathogens which induces formation of both cellular and humoral immunity against the respective microorganism. This property of genetic vaccines is used for the development of prophylactic schemes. New vaccines are currently being designed to prevent a variety of infections. The aim of the present review is to outline major trends in genetic vaccination leading to the improvement of its efficacy.

Virus-like particle vaccine containing hemagglutinin confers protection against 2009 H1N1 pandemic influenza.

Immunization of the world population before an influenza pandemic such as the 2009 H1N1 virus spreads globally is not possible with current vaccine production platforms. New influenza vaccine technologies, such as virus-like-particles (VLPs), offer a promising alternative. Here, we tested the immunogenicity and protective efficacy of a VLP vaccine containing hemagglutinin (HA) and M1 from the 2009 pandemic H1N1 influenza virus (H1N1pdm) in ferrets and compared intramuscular (i.m.) and intranasal (i.n.) routes of immunization. Vaccination of ferrets with VLPs containing the M1 and HA proteins from A/California/04/2009 (H1N1pdm) induced high antibody titers and conferred significant protection against virus challenge. VLP-vaccinated animals lost less weight, shed less virus in nasal washes, and had markedly lower virus titers in all organs tested than naïve controls. A single dose of VLPs, either i.m. or i.n., induced higher levels of antibody than did two doses of commercial split vaccine. Ferrets vaccinated with split vaccine were incompletely protected against challenge; these animals had lower virus titers in olfactory bulbs, tonsils, and intestines, but lost weight and shed virus in nasal washes to a similar extent as naïve controls. Challenge with heterologous A/Brisbane/59/07 (H1N1) virus revealed that the VLPs conferred minimal cross-protection to heterologous infection, as revealed by the lack of reduction in nasal wash and lung virus titers and slightly higher weight loss relative to controls. In summary, these experiments demonstrate the strong immunogenicity and protective efficacy of VLPs compared to the split vaccine and show that i.n. vaccination with VLPs has the potential for highly efficacious vaccination against influenza.

A virus-like particle-based Epstein-Barr virus vaccine.

Epstein-Barr Virus (EBV) is an ubiquitous human herpesvirus which can lead to infectious mononucleosis and different cancers. In immunocompromised individuals, this virus is a major cause for morbidity and mortality. Transplant patients who did not encounter EBV prior to immunosuppression frequently develop EBV-associated malignancies, but a prophylactic EBV vaccination might reduce this risk considerably. Virus-like particles (VLPs) mimic the structure of the parental virus but lack the viral genome. Therefore, VLPs are considered safe and efficient vaccine candidates. We engineered a dedicated producer cell line for EBV-derived VLPs. This cell line contains a genetically modified EBV genome which is devoid of all potential viral oncogenes but provides viral proteins essential for the assembly and release of VLPs via the endosomal sorting complex required for transport (ESCRT). Human B cells readily take up EBV-based VLPs and present viral epitopes in association with HLA molecules to T cells. Consequently, EBV-based VLPs are highly immunogenic and elicit humoral and strong CD8+ and CD4+ T cell responses in vitro and in a preclinical murine model in vivo. Our findings suggest that VLP formulations might be attractive candidates to develop a safe and effective polyvalent vaccine against EBV.

A candidate dual vaccine against influenza and noroviruses.

The extracellular domain of the matrix protein 2 (M2e) of influenza viruses is highly conserved among all influenza A subtypes, making it a suitable target for a universal influenza vaccine. In this study, we demonstrated an enhanced immune response and protection of a chimeric M2e vaccine against influenza A viruses using our newly developed vaccine platform, the norovirus P particle, to present the M2e peptide. The 23-amino acid peptide was inserted into one of the surface loops of the P protein, resulting in 24 copies of M2e presented on each P particle. Significantly (P<0.001) increased antibody responses to M2e were observed in mice immunized with the P particle-M2e chimera compared with those immunized with the free M2e peptides. Mice immunized with the P particle-M2e vaccine were fully protected (100% survived) against lethal challenge of a mouse adapted human influenza virus PR8 (H1N1), while only low survival rates (<12.5%) were found in mice immunized with the free M2e peptides or wild type P particle. In addition, the mouse sera collected after immunization with the P particle-M2e vaccine were able to block the binding of norovirus virus-like particle and P particle to histo-blood group antigen receptors. These results suggest that the P particle-M2e chimera can be used as dual vaccine against both noroviruses and influenza viruses.

Baculovirus-produced influenza virus-like particles in mammalian cells protect mice from lethal influenza challenge.

Influenza virus-like particles (VLPs) are effective vaccines against influenza infection, which can be produced either in insect cells by recombinant baculovirus (BV) infection or in mammalian cells by DNA plasmid transfection. However, VLPs produced from baculovirus/insect cells are difficult to purify due to baculovirus contamination; VLPs produced by plasmid transfection are limited by scale-up capability. In this study, a BacMam BV, in which three CMV-promoters drive the hemagglutinin, neuraminidase, and matrix of influenza virus was constructed. This baculovirus can deliver these genes into mammalian cells/hosts and subsequently influenza VLPs can be produced and secreted from transduced cells. Transduction conditions were optimized and influenza VLPs were purified from transduced 293T cells. Mice were vaccinated with BV transduction-produced VLPs, plasmid transfection-produced VLPs, and BacMam BV. Two vaccinations of each vaccine induced high hemagglutination-inhibition (HAI) titers and prevented influenza virus infection. In contrast, following a single vaccination, all mice vaccinated with each vaccine had significantly lower lung viral titers compared to unvaccinated mice. Remarkably, mice vaccinated with a single dose of BV transduction-produced VLPs survived challenge, whereas mice vaccinated with one dose of BacMam BV- or plasmid transfection-produced VLPs had 60-80% survival. This finding is particularly significant for producing easily purified VLPs. The BacMam system is an alternative strategy for VLP production, which is easy to scale up and purify. Besides, BacMam BV can be used as a gene delivery vector to produce VLPs in vivo, to stimulate immune responses.

Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV.

SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 µg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 µg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents.